Methods for cannabinoid quantification

ABSTRACT

A method for quantification of the concentration of one or more cannabinoid compounds in a liquid sample is provided. The method involves contacting the liquid sample with at least one cannabinoid-sensitive visualization reagent, allowing the at least one cannabinoid-sensitive visualization reagent to develop for a defined amount of time; and comparing the resulting color change of the at least one cannabinoid-sensitive visualization reagent to a calibrated quantification reference chart.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application No.16/825,570, filed on Mar. 20, 2020, now allowed, which is a continuationapplication of U.S. Non-Provisional application Ser. No. 15/863,506,filed Jan. 5, 2018, entitled “Methods for Cannabinoid Quantification”nowU.S. Pat. No. 10,634,689, which is a continuation of U.S.Non-Provisional application Ser. No. 15/587,162, filed May 4, 2017,entitled “Methods for Cannabinoid Quantification” now abandoned, whichis a continuation application of U.S. Non-Provisional application Ser.No. 14/771,592, filed Aug. 31, 2015, entitled “Methods for CannabinoidQuantification” now abandoned, which is a U.S. National Stage EntryApplication of International Application Serial NumberPCT/CA2014/000157, filed Feb. 28, 2014, entitled “Methods forCannabinoid Quantification”, which claims priority to U.S. ProvisionalPatent Application Ser. No. 61/771,263, filed Mar. 1, 2013, entitled“Cannabinoid Quantification”, U.S. Provisional Patent Application Ser.No. 61/827,128, filed May 24, 2013 entitled “Cannabinoid Quantification,In Solution” and, U.S. Provisional Patent Application Ser. No.61/884,409, filed Sep. 30, 2013, entitled “Cannabinoid Quantification,”all of which are hereby incorporated by reference for all purposes.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for accuratequantification of cannabinoid compounds in a sample.

BACKGROUND OF THE INVENTION

The legal production, sale and use of cannabis for medical purposes isbecoming more prevalent in many countries, including the United Statesand Canada. It is important for medical cannabis growers, dispensariesand end-users to know the concentration of certain cannabinoids,particularly delta-9-tetrahydrocannabinol (THC) in specific plantsamples—such information can be important for strain development, foroptimizing production/growing cycles, for complying withjurisdiction-specific legal requirements, and for other quality controlpurposes.

Quantification of cannabinoids in a sample generally requires alaboratory test, for instance using gas chromatography, which can beexpensive and generally requires time to send the sample to anappropriate lab and wait for the results. Home-based tests, such asAlpha-Cat, have been developed using thin-layer chromatography, howeverthis test is still quite complex for the average untrained user, and thequantification resolution of the test is limited.

Reagents are known in the art that react chemically with CBD, underspecific reaction conditions, to create a color change. Mechoulam(Tetrahedron 24(16): 5615-5624, 1968) teaches that CBD, when contactedwith 5% ethanolic potassium hydroxide (KOH) is converted to quinone,which has a purple color.

U.S. Pat. No. 4,771,005, issued to Spiro, teaches methods forpositive/negative cannabinoid detection in human sample using adiazonium salt, for instance Fast Blue BB.

WO/1989/009395 (published in the name of Fraser and Johnson) teaches atest paper for positive/negative cannabinoid detection using diazoniumsalt, for instance Fast Blue BB.

U.S. Pat. No. 8,124,420, issued in the name of Amisar, teaches a testpaper and kit for detection and/or identification of a range of drugs ofabuse, which may include cannabinoids, and the test paper may include adiazonium salt, for instance Fast Corinth V.

Fischedick et al. (2009 Phytochem Anal 20:421-6.) teaches a method forquantifying cannabinoids in a sample using high performance thin layerchromatography (HPTLC).

SUMMARY OF THE INVENTION

In one aspect of the invention, a method for quantification of theconcentration of one or more cannabinoid compounds in a liquid sample isprovided. The method involves contacting the liquid sample with at leastone cannabinoid-sensitive visualization reagent; allowing the at leastone cannabinoid-sensitive visualization reagent to develop for a definedamount of time; and comparing the resulting color change of the at leastone cannabinoid-sensitive visualization reagent to a calibratedquantification reference chart. The at least one cannabinoid-sensitivevisualization reagent may be present in a liquid form. The at least onecannabinoid-sensitive visualization reagent may be present in a solidform. The liquid sample may be contacted separately with the at leastone cannabinoid-sensitive visualization reagent. The at least onecannabinoid-sensitive visualization reagent may be impregnated in a teststrip. The test strip may include a porous matrix uniformly impregnatedwith the at least one cannabinoid-sensitive visualization reagent. Theat least one cannabinoid-sensitive visualization reagent reacts withcannabinoids, which can include THC, CBD, or CBN. Optionally, thecannabinoid may be solely THC, CBD or another cannabinoid. The at leastone cannabinoid-sensitive visualization reagent may include one or morediazonium salts, a Duquenois reagent, a Ghamrawy reagent, or a modifiedGhamrawy reagent. The one or more diazonium salts may include Fast BlueB, Fast Blue BB, Fast Red B, Fast Red GG, Fast Orange GR, Fast CorinthV, Fast Garnet GC, Fast Red AV, or Fast Bordeaux GP. Optionally, the atleast one cannabinoid-sensitive visualization reagent may be a strongbase, which may be potassium hydroxide or sodium hydroxide. Further, theat least one cannabinoid-sensitive visualization reagent may beimpregnated in a test strip. Further still, the test strip may include aporous matrix uniformly impregnated with the at least onecannabinoid-sensitive visualization reagent.

In another aspect of the invention, a method for quantification of oneor more cannabinoid compounds in a solid test sample is provided. Themethod involves contacting the solid test sample with an extractionsolvent, wherein one or more cannabinoids are extracted from the solidsample into an extraction solvent resulting in a cannabinoid-containingliquid extraction solution; contacting the cannabinoid-containing liquidextraction solution with one or more cannabinoid-sensitive visualizationreagents; and comparing the intensity of the resulting color change ofthe one or more cannabinoid-sensitive visualization reagents to acalibrated quantification reference chart.

In another aspect of the invention, an apparatus which includes a teststrip impregnated with at least one cannabinoid-sensitive visualizationreagent is disclosed. Optionally the at least one cannabinoid-sensitivevisualization reagent may be one or more diazonium salts, a Duquenoisreagent, a Ghamrawy reagent, a modified Ghamrawy reagent, and potassiumhydroxide.

In another aspect of the invention, a kit for quantification of theconcentration of one or more cannabinoid compounds in a sample isdisclosed. The kit includes at least one cannabinoid sensitivevisualization reagent, and a calibrated quantification reference chart.Optionally, the at least one cannabinoid-sensitive visualization reagentis present in a liquid form or a solid form. Further and optionally, theat least one cannabinoid-sensitive visualization reagent is pre-measuredas a dry reagent in one or more reaction containers. Further still, theat least one cannabinoid-sensitive visualization reagent is impregnatedinto a test strip.

Aspects of the present invention are based, in part, on the finding thatcertain cannabinoid-sensitive visualization reagents may be used toquantify the cannabinoid concentration in a sample. Herein, it has beendemonstrated that the use of one or more cannabinoid-sensitivevisualization reagents which cause an absorbance shift (color change) ina solution upon contact with cannabinoids including THC and/or CBD, maybe utilized to quantify the concentration of such cannabinoids in agiven sample. Quantification is achieved by contacting the one or morecannabinoid-sensitive visualization reagents with a liquidcannabinoid-containing sample, or alternatively with a liquid extractionfrom a solid cannabinoid-containing sample, and comparing the resultingabsorbance shift to that caused by samples with known cannabinoidconcentrations. Herein, examples have been provided of such assays todetermine the concentration of THC, CBD, and/or other cannabinoids in asolid plant test sample, or a liquid test sample. Examples have beenprovided of a calibrated quantification reference chart, useful in saidassay, calibrated to determine the cannabinoid concentration in a solidplant test sample, and suitable for solid plant test samples havingcannabinoid concentration ideally between 0-25%, but potentially higher.In some embodiments, the calibrated quantification reference chart hasbeen optimized for this cannabinoid concentration range by extractingcannabinoids from series of solid samples having known cannabinoidconcentrations ranging from 0% to 25%, using a consistent and uniformextraction solvent composition, volume, and extraction time for all ofthe samples, and contacting the resulting cannabinoid-containingextraction liquid with a defined amount of one or morecannabinoid-sensitive visualization reagents. The resulting absorbanceshift, or color change, caused by each of the samples of knowncannabinoid concentration provides a calibrated quantification referencechart for use in the experimental assay for the test sample—byextracting the cannabinoids from the unknown test sample using the exactsame extraction solvent composition, volume, and extraction time;contacting the resulting cannabinoid-containing extraction liquid withthe same amount/concentration of the one or more cannabinoid-sensitivevisualization reagents; and comparing the resulting absorbance shift(s),or color change(s), with the calibrated quantification reference chart,one may thus determine the cannabinoid concentration in the unknownsolid test sample. In the examples provided herein, the extractionsolvent composition, volume, and extraction time have been optimized toensure that the absorbance shift caused by contacting the resultingcannabinoid-containing extraction solution with the one or morecannabinoid-sensitive visualization reagents is in the optimized rangeof the visualization reagent, such that the absorbance, or color changeintensity, is proportional to the cannabinoid concentration in the testsample. In certain aspects of the invention, the cannabinoid that may bequantified is one or more of THC, CBD and/or CBN, and thecannabinoid-sensitive visualization reagent one or more of: i) diazoniumsalt, for instance Fast Blue B, Fast Blue BB, Fast Blue RR, Fast CorinthV, Fast Garnet, Fast Bordeaux, and the like; ii) a Duquenois reagent;and iii) a Ghamrawy reagent or modified Ghamrawy reagent. In certainaspects of the invention, the cannabinoid that may be quantified is CBD,and the cannabinoid-sensitive visualization reagent is a strong base forinstance potassium hydroxide or sodium hydroxide, wherein the medium inwhich the strong base contacts the CBD is methanol, ethanol, propanol,or another lower alcohol solvent.

Examples have been provided of a variety of visualization reactioncompositions and methods suitable for the present invention. In certainaspects of the invention, the visualization reaction may occur in aliquid solution, wherein the one or cannabinoids of the liquid testsample, or extracted from the solid test sample, are contacted with theone or more cannabinoid-sensitive visualization reagents in solution,for instance using a solvent composition that is permissive for thevisualization reaction. In certain aspects, the cannabinoids of the testsample are separately contacted with each of the one or morecannabinoid-sensitive visualization reagents in separate reactionvessel. In other aspects of the invention, the visualization reactionmay be accomplished using a test paper, wherein the test paper isimpregnated with a cannabinoid-sensitive visualization reagent. Incertain aspects, the test paper may be a test strip comprising one ormore test pads, wherein each test pad is impregnated with a differentcannabinoid-sensitive visualization reagent. In this regard, one ofunique features of the present invention is based in part on the findingthat certain cannabinoid-sensitive visualization reagents havedifferent, and potentially overlapping, quantification ranges and/orsensitivities. Thus, in certain aspects of the present invention, theuse of more than one cannabinoid-sensitive visualization reagent mayallow for an extended range and accuracy of quantification. Anotherfeature is that certain cannabinoid sensitive visualization reagents maybe impregnated on a test strip and included in a kit in such a way thatthe activity of the visualization reagent is retained when the strip iseventually used by the end-user.

BRIEF DESCRIPTION OF THE FIGURES

Two sets of identical figures are being submitted herewith: a first setrendered in black and white, and a second set is rendered in colour.

FIG. 1 depicts a calibrated quantification reference chart for THCsamples having an ideal range of 5-20%. The quantification referencechart was generated using Fast Blue BB. The chart shows a light yellowcolour at the left side, changing to a darker yellow/orange on the rightside.

FIG. 2 depicts an extended range calibrated quantification referencechart. The chart includes overlapping ranges for 2 differentcannabinoid-sensitive visualization reagents, to allow forquantification from 0% to over 25% THC in a solid sample.

FIG. 3 depicts a calibrated quantification reference chart for CBDsamples having ideal range 0-15%. Moving from left to right, the figureshows the color changing from very light purple at the lower end of thescale to a much darker purple at the higher end of the scale.

FIG. 4 depicts a calibrated reference chart for Fast BB impregnated teststrips. The chart shows that, moving from left to right, the colorchange of the strip goes from a very light orange/red at the low end ofthe scale to a much darker red at the higher end of the scale.

FIG. 5 depicts representative test papers prepared using 30% KOH (left),10% KOH (middle), and 1% KOH (right).

FIG. 6 depicts a CBD Calibrated Reference Chart. Moving from left toright on the chart, the color change shown on the chart goes from a verylight violet/purple for the lower CBD % to a much darker purple forhigher CBD %.

FIG. 7 depicts a THC Calibrated Reference Chart using modified Ghamrawyreagent impregnated test papers. Moving from left to right on the chart,the color change shown on the chart goes from a very light violet/purplefor the lower THC % to a much darker purple for higher THC %.

DETAILED DESCRIPTION

Any terms not directly defined herein shall be understood to have themeanings commonly associated with them as understood within the art ofthe invention. As employed throughout the specification, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings.

The term ‘sample’ means a complex substance that may be tested oranalyzed for the presence of certain compounds. A ‘sample’ may be aliquid sample or a solid sample. A liquid sample may comprise a bodilyfluid such as urine or blood. A liquid sample may comprise a solventextract of a solid sample, wherein various compounds from the solidsample are extracted into the liquid solvent using methods known in theart. A solid sample may include plant material, for instance cannabisplant material.

The term ‘cannabis’ means a genus of flowering plants that includesthree putative species, Cannabis sativa, Cannabis indica, and Cannabisruderalis. The term ‘cannabis’ may also refer to plant material derivedor extracted from the cannabis plant, for instance the leaves, stem,seeds, flowering bodies, or other portions of the plant.

The term ‘cannabinoid’ or ‘cannabinoids’ means a class of chemicalcompounds which include the phytocannabinoids (oxygen-containing C21aromatic hydrocarbon compounds found in the cannabis plant), andchemical compounds which mimic the actions of phytocannabinoids or havea similar structure (e.g., endocannabinoids, found in the nervous andimmune systems of animals and that activate cannabinoid receptors).Phytocannabinoids are known to occur in significant quantities in thecannabis plant, and may include, but are not limited totetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), andcannabigerol (CBG).

The term ‘THC’ means tetrahydrocannabinol and may include differentisoforms and variants, such as delta-9-Tetrahydrocannabinol (Δ9-THC) anddelta-8-tetrahydrocannabinol (Δ8-THC). The inventors herein disclosemethods and an apparatus for quantification of THC and/or othercannabinoids from a sample, for instance from a solid cannabis sample.

The term ‘CBD’ means cannabidiol, a cannabinoid often found in cannabis,and having a CAS registry number 13956-29-1. Cannabidiol is known tohave many beneficial medicinal qualities.

The term ‘extraction’ means to transfer compounds from a sample intoanother medium, for instance into a liquid solvent. The solvent may bechosen such that certain desired compounds are soluble in the solvent,and thus when the sample is contacted with the solvent, the desiredcompounds are transferred to the solvent. Extraction of solid samplesmay be aided or enhanced by grinding, macerating or otherwisepulverizing the sample material. Extraction is aided by shaking,vortexing or otherwise mixing the solvent with the sample. Uniformextraction yields may be achieved by using defined extraction methods,including duration of extraction, solvent composition, and the like. Incertain embodiments, a solid sample may be heated prior toextraction—this may serve to chemically convert certain compounds thatare not soluble in the extraction solvent into compounds that aresoluble in the extraction solvent. Furthermore, the converted compoundsmay be more reactive with visualization reagents. In certain embodimentsof the invention, solid cannabis samples are heated prior to extractionin order to decarboxylate the acid form of THC, CBD, etc. into non-acidforms which are more readily extracted and more readily react with thevisualization reagents of the present invention. Ideally, the heatconversion is carried out a temperature that is below the volatilizationtemperature of the cannabinoids, but above the decarboxylationtemperature. In this way, any THC-A and CBD-A in the solid sample, maybe converted to THC and CBD prior to extraction, and the subsequentvisualization reactions of the invention will give a more accuratequantification of total THC and/or total CBD in the sample.

A ‘visualization reagent’ means a reagent or compound that changes colorupon contact with a particular analyte or class of analytes, oralternatively causes the analyte to change color upon contact with thevisualization reagent, or alternatively causes a color change in areaction medium containing a particular analyte or class of analytes.The change in color will result in a change in the spectral absorbanceof the reaction medium, detection of which may be visible to the nakedeye, or may be more accurately quantified using a device such as acolorimeter, spectrometer, spectrophotometer, or the like. Visualizationreagents may alternatively be referred to as colorimetric reagents.There are numerous types of visualization reagents described in the art.Certain visualization reagents may be suitable for quantification ofanalytes, while others may not. Suitable visualization reagents maycause a color change that is proportional to the concentration of theanalyte, over a specific analyte concentration range—sometimes referredto as the ‘linear range’ or ‘optimal range’ of the visualizationreagent. The optimal range of the visualization reagent for the specificanalyte must be wide enough to provide quantification information acrossa sufficient range to be a useful visualization reagent. A visualizationreaction may require contacting the visualization reagent with theanalyte in reaction conditions suitable to cause the appropriatechemical reaction. For instance, a suitable solvent may be used tofacilitate the visualization reaction. The suitable solvent of thepresent invention may be an alcohol such as methanol, ethanol, propanoland the like.

A ‘diazonium salt’, or ‘diazonium compound’, is a compound belonging toa group of organic compounds sharing a common functional group R—N2+ X—where R can be any organic residue such alkyl or aryl and X is aninorganic or organic anion such as a halogen. Diazonium salts,especially those where R is an aryl group, are important intermediatesin the organic synthesis of azo dyes. Diazonium salts are often used asvisualization reagents by conversion of the diazonium salt into an azodye, such conversion causing a change in absorbance. Examples ofdiazonium salts may include, but are not limited to, Fast Blue B(3,3′-dimethoxybenzidine (o-dianisidine)), Fast Blue BB(4-Benzoylamino-2,5-diethoxyaniline), Fast Red B (2-Methoxynitroaniline), Fast Red GG (4-Nitroaniline), Fast Orange GR(2-Nitroaniline), Fast Corinth V(2-methoxy-5-methyl-4-(4-methyl-2-nitrophenyl)-azobenzene-diazonium),Fast Garnet GC (4-(m-Tolylazo)-3-methylaniline), Fast Red AV, and FastBordeaux GP.

A ‘Duquenois reagent’ is a reagent used in a Duquenois-Levine test fordetecting cannabis (see: P. Duquenois and H. N. Moustapha, J. Egypt.Med. Ass., 1938, 21, 224.) A Duquenois reagent may comprise a mixture ofvanillin and acetaldehyde, along with hydrochloric acid. In certainembodiments, the hydrochloric acid may be substituted for another acid.In certain embodiments, the strong acid may be present as a dry form,for instance p-toluenesulfonic acid, and may be impregnated in a testpaper/strip.

A ‘Ghamrawy reagent’ refers to a combination of compounds that may beused for detection of THC and CBD. A Ghamrawy reagent is furtherdescribed in: Kovar, Karl-Artur and Martina Laudszun. (Chemistry andReaction Mechanisms of Rapid Tests for Drugs of Abuse and PrecursorsChemicals, United Nations Scientific and Technical Notes v. 89-51669,Germany. February 1989. The Ghamrawy reagent consists ofp-dimethylaminobenzaldehyde (p-DMAB) along with concentrated sulfuric orhydrochloric acid. In certain embodiments, the Ghamrawy reagent may bemodified—the inventors have identified other strong acids that may beuseful for catalyzing the color reaction with p-DMAB. For instance,p-toluenesulfonic acid that may be used instead of hydrochloric acid orsulfuric acid. This acid is advantageous for use in test strips, byvirtue of the fact that p-toluenesulfonic acid may be present as asolid, whereas hydrochloric and sulfuric acid generally are not.Furthermore p-toluenesulfonic acid is non-corrosive and thus will notdegrade the test strip. This surprising finding has enabled the use oftest papers/strips impregnated with the modified Ghamrawy reagent forquantification of THC and/or CBD. Thus, a ‘modified Ghamrawy reagent’comprises p-DMAB and a strong acid, for instance p-toluenesulfonic acid.Other strong acids that may be present as a solid may also be used in amodified Ghamrawy reagent.

The term ‘KOH’ means ‘potassium hydroxide’. Potassium hydroxide may havethe CAS registry number 1310-58-3. The term ‘NaOH’ means ‘sodiumhydroxide. Sodium hydroxide may have the CAS registry number 1310-73-2.

The term ‘cannabinoid-sensitive visualization reagent’ refers to areagent such as a visualization reagent that undergoes a change inproperties, such as spectral absorbance, upon contact with acannabinoid. In the context of the present invention, the change inchemical properties may occur due to a change in either reactant in thevisualization reaction, or even a change in the reaction medium itselfAlternatively, the change in chemical properties may occur due to theformation of a new compound, such as a reaction by-product. Acannabinoid-sensitive visualization reagent may be a diazonium salt.Certain diazonium salts are known to cause a change of color in thepresence of cannabinoids such as THC, CBD and/or CBN—such diazoniumsalts include, but are not limited to, Fast Blue B, Fast Blue BB, FastCorinth V, and Fast Garnet GC. A cannabinoid-sensitive visualizationreagent may be a Duquenois reagent. A cannabinoid-sensitivevisualization reagent may be a Ghamrawy reagent or a modified Ghamrawyreagent. A cannabinoid-sensitive visualization reagent may be potassiumhydroxide or sodium hydroxide.

The term ‘permissive’ or ‘permissive solvent’ refers to solvents thathave a composition that allow the desired chemical reaction to occur.For instance, certain chemical reactions may proceed much more readilyin an alcohol than in water. In such case, the alcohol would be apermissive solvent and would be the desired solvent for the reaction. Invarious embodiments, the use of permissive solvents is important for theutility of the invention.

The term ‘calibrated quantification reference chart’ means a chart,graph, or other visual representation showing the specific absorbanceshift or color change of one or more specific visualization reagentscaused by a specific analyte across a range of specific concentration,under specific conditions, such that comparison of an analyte at anunknown concentration, using the same one or more specific visualizationreagents under the same specific conditions, to the calibratedquantification reference chart will allow one to determine theconcentration of that analyte in the unknown test sample. The specificabsorbance shift or color change may be determined for instance using atest paper, test strip, a colorimeter, a spectrometer, or by visualinspection. The present invention utilizes a calibrated quantificationreference chart to determine the concentration of cannabinoids in asample. In certain embodiments of the invention, a calibratedquantification reference chart may be used or created for determiningthe cannabinoid concentration in an unknown solid sample by extractingcannabinoids from series of solid samples having known cannabinoidconcentrations, using a uniform extraction solvent composition, volume,and extraction time for all of the samples, and contacting the resultingcannabinoid-containing extraction liquid with a defined amount of one ormore cannabinoid-sensitive visualization reagents. The resultingabsorbance shift(s), or color change(s), caused by each of the samplesof known cannabinoid concentration provides a calibrated quantificationreference chart for use in the experimental assay for the unknown testsample. By then extracting the cannabinoids from the unknown test sampleusing the exact same extraction solvent composition, volume, andextraction time; contacting the resulting cannabinoid-containingextraction liquid with the same amount/concentration of one or morecannabinoid-sensitive visualization reagents; and comparing theresulting absorbance shift(s), or color change(s), with the calibratedquantification reference chart, one may thus determine the cannabinoidconcentration in the unknown solid sample.

The term ‘porous matrix’ refers to a solid material, ie. a matrix, thatis permeated with pores or small holes to allow absorbance of a fluidinto the matrix. A porous matrix may be a type of paper or filter, suchas blotting paper. Examples of a porous matrix include Whatman paper,CF1, CF2, CF3, cellulose paper, and the like.

The term ‘test paper’ refers to a porous matrix which is impregnatedwith a diagnostic reagent, for instance a cannabinoid-sensitivevisualization reagent such as a diazonium salt, Duquenois reagent,Ghamrawy reagent, or modified Ghamrawy reagent. Impregnation of theporous matrix with the reagent may be accomplished in several differentways. The reagent to be impregnated into the porous matrix to form thetest paper may first be dissolved in a suitable solvent, and the porousmatrix then contacted with or submersed in the resulting solutioncontaining the dissolved reagent. For instance, the solvent may be analcohol such as methanol, ethanol or propanol. Preferably, the solventmay be a ketone such as acetone or methyl ethyl ketone—these solvent mayprevent degradation of the dye and reduce background coloration on thetest strips. Evaporation of the solvent will result in a dry test stripimpregnated with the reagent. Suitable solvents should thus be chosensuch that the reagent is miscible in the solvent, and such that thesolvent is volatile enough to effectively evaporate away from the porousmatrix. Examples of suitable solvents include, but are not limited to,water, methanol, ethanol, isopropanol, petroleum ether, methyl ethylketone, acetone, dimethylchloride, hexane. In certain embodiments, thevisualization reagent may be heated after impregnation of the testpaper, or during the drying of the test paper. Such heating may activatethe visualization reagent. In certain embodiments the test paper may besubsequently sealed into an air tight package, for instance by vacuumsealing. This may preserve the activation or activity of thevisualization reagent. A test paper may be contacted with a testcompound, for instance a cannabinoid, in a suitable solvent, and thereaction of the test compound with the diagnostic reagent may cause thetest paper to change color. In various embodiments of the invention, thecolor change of the test paper may be proportional to the concentrationof one or more cannabinoids in the solution, and may thus be used toquantify the cannabinoid concentration.

The term ‘test strip’ refers to a strip of material to which is attachedone or more test papers. A test strip may be elongated to allow for easeof contacting with the test solution, for instance by dipping into atest tube or the like. The test strip may include i) a backing material,which may be absorbent or non-absorbent, and is preferentially but notnecessarily inert; ii) one or more test papers comprising one or morecannabinoid-sensitive visualization reagents; and iii) an adhesive toattach the one or more test papers to the backing material.

Quantification of cannabinoid concentration in liquid or solid samplesusing one or more cannabinoid-sensitive visualization reagents.

Herein, the inventors describe methods for determination of thecannabinoid concentration in liquid or solid samples using one or morecannabinoid-sensitive visualization reagents. The inventors havesurprisingly determined that certain cannabinoid-sensitive visualizationreagents can be utilized in conjunction with a calibrated referencechart to determine the actual concentration of cannabinoids in a sample,rather than merely detecting the presence or absence of cannabinoids,and are thus suitable for cannabinoid quantification. In certainembodiments of the invention, such one or more cannabinoid-sensitivevisualization reagents are first contacted with a cannabinoid-containingliquid sample, or with a liquid extraction of a cannabinoid-containingsolid sample, under conditions which cause a color change or absorbanceshift of the one or more visualization reagents, wherein such colorchange or absorbance shift is proportional to the cannabinoidconcentration. The color change or absorbance shift of the visualizationreagent is then compared to a calibrated quantification reference chart,wherein the calibrated quantification reference chart is created bytesting a series of liquid or solid sample of known cannabinoidconcentration under the exact same test conditions as the sample ofunknown concentration—ideally the calibrated quantification referencechart would show the color change or absorbance shift at a number ofknown cannabinoid concentrations. In such a way, one may compare thecolor change or absorbance shift of the sample of unknown cannabinoidconcentration to the calibrated quantification reference chart todetermine the cannabinoid concentration in the unknown sample.Determination of the color change or absorbance shift may beaccomplished by numerous means, for instance using a colorimeter orspectrophotometer, or by visual inspection. In certain embodiments, theone or more cannabinoid-sensitive visualization reagents is a diazoniumsalt, such as Fast Blue B, Fast Blue BB, Fast Corinth V, Fast Garnet GC,and the like, which are useful for quantification of THC, CBD and/orCBN. In other embodiments, the cannabinoid-sensitive visualizationreagent is a strong base, for instance potassium hydroxide or sodiumhydroxide, useful for quantification of CBD. In other embodiments, theone or more cannabinoid-sensitive visualization reagents is a Duquenoisreagent, a Ghamrawy reagent, or a modified Ghamrawy reagent.

In one embodiment of the invention, there is provided a method forquantification of the concentration of one or more cannabinoid compoundsfrom a liquid test sample, the method involves: 1) contacting thecannabinoid-containing liquid sample with a defined amount of one ormore cannabinoid-sensitive visualization reagents; 2) allowing theresulting visualization reaction(s) to develop for a defined amount oftime; and 3) comparing the intensity of the resulting color change, orabsorbance shift, of the one or more cannabinoid-sensitive visualizationreagents to a calibrated quantification reference chart, wherein suchcomparison allows determination of the cannabinoid concentration in thetest sample. The calibrated quantification reference chart may beproduced, for instance, by contacting a series of calibrated liquidsamples having pre-determined cannabinoid concentrations with the sameamount/composition of one or more cannabinoid-sensitive visualizationreagents under the exact same conditions as to be used for the unknowntest sample, including using the same one or morecannabinoid-visualization reagents and the same color development timeas to be used for the test sample. In certain embodiments wherein two ormore cannabinoid-sensitive visualization reagents are used, eachvisualization reagent may be separately contacted with thecannabinoid(s) of the test sample. In certain embodiments, the methodmay be useful for the quantification of cannabinoids THC, CBD and/or CBNin a liquid sample. The one or more cannabinoid-sensitive visualizationreagents may be a diazonium salt. The one or more cannabinoid-sensitivevisualization reagents may be chosen from the following: Fast Blue B,Fast Blue BB, Fast Blue RR, Fast Corinth V, Fast Garnet, Fast Bordeaux.The diazonium salt(s) and the cannabinoids may be contacted inpermissive solvent. The one or more cannabinoid-sensitive visualizationreagents may be a Duquenois reagent, a Ghamrawy reagent, and/or amodified Ghamrawy reagent. In other embodiments, the method may beuseful for the quantification of CBD in a liquid sample. Thecannabinoid-sensitive visualization reagent may be a strong base, forinstance potassium hydroxide, sodium hydroxide. The strong base, forinstance potassium hydroxide, and/or cannabinoid(s) may be contacted ina permissive solvent such as a lower alcohol, for instance methanol,ethanol, propanol, and the like.

In another embodiment of the invention, there is provided a method forquantification of one or more cannabinoid compounds in a solid testsample, the method involves: 1) contacting a defined amount of solidtest sample with a defined volume of an extraction solvent for a definedamount of time, wherein one or more cannabinoids are extracted from thesolid sample into an extraction solvent resulting in acannabinoid-containing liquid extraction solution; 2) contacting theresulting cannabinoid-containing liquid extraction solution with definedamount of one or more cannabinoid-sensitive visualization reagent; 3)allowing the resulting visualization reaction(s) to develop for adefined amount of time; and 4) comparing the intensity of the resultingcolor change, or absorbance shift, of the one or morecannabinoid-sensitive visualization reagents to a calibratedquantification reference chart, wherein such comparison allowsdetermination of the cannabinoid concentration in the solid test sample.The calibrated quantification reference chart may be produced byperforming the same assay method on a series of solid samples havingknown cannabinoid concentrations and recording the resulting absorbancechanges. For instance, the calibrated quantification reference chart maybe produced by contacting a series of solid samples havingpre-determined cannabinoid concentrations with the same defined amountof the same defined extraction solvent composition, and then contactinga defined amount of resulting cannabinoid-containing extraction solutionwith one or more cannabinoid-sensitive visualization reagents under theexact same conditions as to be used for each test sample, includingusing the same one or more cannabinoid-visualization reagentcompositions and concentrations, and the same color development time asto be used for the test sample. In certain embodiments wherein two ormore cannabinoid-sensitive visualization reagents are used, eachvisualization reagent is separately contacted with the cannabinoid(s) ofthe test sample. In certain embodiments, the method may be useful forquantification of the concentration cannabinoids THC, CBD and/or CBN ina solid test sample. The one or more cannabinoid-sensitive visualizationreagent may be a diazonium salt. The cannabinoid-sensitive visualizationreagent may be chosen from the following: Fast Blue B, Fast Blue BB,Fast Blue RR, Fast Corinth V, Fast Garnet, Fast Bordeaux. The diazoniumsalt(s) and the cannabinoids may be contacted in permissive solvent. Thecannabinoid-sensitive visualization reagent may be a Duquenois reagent,a Ghamrawy reagent, or a modified Ghamrawy reagent. In certainembodiments, the method may be useful for the quantification of CBD in asolid test sample. The cannabinoid-sensitive visualization reagent maybe a strong base, for instance potassium hydroxide, sodium hydroxide.The strong base, for instance potassium hydroxide, and/or cannabinoid(s)may be contacted in a permissive solvent such as a lower alcohol, forinstance methanol, ethanol, propanol, isopropanol, etc.

Extended Range of Quantification

Herein, cannabinoid quantification assays are described which have anextended range of quantification. The cannabinoid quantification assaysdescribed in various embodiments of the invention all have a lower andupper limit of quantification, below and above which quantification isrelatively ineffective. Below the lower limit of quantification, thecannabinoid concentration is too low to cause a significant absorbanceshift, or color change, from the cannabinoid-sensitive visualizationreagent. Above the upper limit of quantification, the absorbance shift,or color change, may become saturated such that no further color changemay be detected, even with an increased concentration of the testanalyte. Optimal quantification results are thus achieved between thelower and upper limits of quantification, and the greater the distancebetween these two values, the more useful the assay may be—suitablevisualization reagents must be tested and selected in order to obtain auseful quantification assay. Since cannabinoids may exist at wide rangedifferent concentrations in a particular sample, it would be desirableto develop assays with extended ranges. In order to accomplish this, thepresent inventors take advantage of the fact that differentvisualization reagents, such as cannabinoid-sensitive visualizationreagents, often have different sensitivity to, and/or optimal rangesfor, the test compound in question, for instance cannabinoids. Thesedifferent visualization reagents may have different lower and upperlimits of quantification, and thus different effective quantificationranges. In fact, certain visualization reagents may have overlappingquantification ranges, which may be useful for developing an extendedrange assay method using multiple visualization reagents.

In certain embodiments of the invention, there is provided an extendedrange cannabinoid quantification assay method involving the use of twoor more cannabinoid-sensitive visualization reagents, useful forquantification of cannabinoids in a liquid or solid test sample. Theextended range cannabinoid quantification assay method may have certainadvantages over assay methods using a single visualization reagent. Theassay method may be carried out using the same steps as described inother aspects of the invention that use a single visualization reagent(described herein), except that two or more visualization reactions arecarried out in parallel, for instance in separate tubes or on separatetest papers, and the absorbance shift, or color change, of each of thevisualization reactions is compared to a calibrated reference chart,wherein the calibrated reference chart shows the calibratedquantification values for each of the two or more cannabinoid-sensitivevisualization reagents used in the assay. In certain embodiments of theinvention, the assay method may be useful for quantification ofcannabinoid concentrations across an extended range. In certainembodiments of the invention, the two or more cannabinoid-sensitivevisualization reagents have differing cannabinoid sensitivity and oroptimal quantification ranges. In certain embodiments of the invention,the optimal quantification ranges of each of the one or morevisualization reagents are overlapping. The overlapping ranges allowsfor the quantification of cannabinoids in a sample across a wider range,which may improve the utility of the assay. For instance, and by way ofnon-limiting example, user may have a number of solid samples withexpected THC %'s between 0 and 25%; the use of first singlevisualization reagent may allow optimal quantification of THC between0-10% in the solid sample. The use of a second visualization reagentwith an optimal quantification range of 10-25% would extend the overalloptimal range of the assay to 0-25%. The use of additionalcannabinoid-sensitive visualization reagents having even differentquantification ranges may be used to further extend the quantificationrange of the assay method. It should be noted that the use of more thanone visualization reagent can also improve the accuracy ofquantification of cannabinoids in the overlapping region of the optimalquantification ranges of the visualization reagents, due to multiplereadouts from the two or more visualization reagents. In certainembodiments of the invention, the two or more cannabinoid-sensitivevisualization reagents may be diazonium salts and/or Duquenois-Levinereagent and/or Ghamrawy reagent and/or modified Ghamrawy reagent. Incertain embodiments of the invention, the diazonium salts may beselected from: Fast Blue B, Fast Blue BB, Fast Blue RR, Fast Corinth V,Fast Garnet, Fast Bordeaux, or other cannabinoid-sensitive diazoniumsalts.

Visualization

In certain embodiments of the invention, methods are described forcontacting cannabinoids from a test sample with one or morecannabinoid-sensitive visualization reagents, which results in anabsorbance shift, or color change, suitable for quantification of thecannabinoids in the test sample. As exemplified in various workingexamples provided herein, the invention provides different compositionsand methods useful for carrying out the visualization reaction. Incertain embodiments, the visualization reaction may be carried out in aliquid solution, for instance in a reaction vessel such as a test tube,microtube, sample container, etc. A liquid solution of thecannabinoid-sensitive visualization reagent may be added to a liquidcannabinoid-containing solution, according to the methods of the presentinvention, and the color reaction may thus occur in solution. In certainembodiments of the invention, the cannabinoid-sensitive visualizationreagent solution may be prepared fresh at the time of use.

An aspect of the present invention is to to provide simple assays forcannabinoid quantification, and thus there are herein providedadditional methods for reducing the number of required steps to completethe disclosed assays. These simplified assays are thus easier to use,have reduced handling which can increase accuracy, and are more costeffective. In certain cases, the simplification of the assays makes themmore suitable for developing a test kit, as the modified steps mayincrease the shelf life of the reagents in the test kit. For instance,it is known that diazonium salt dyes such as Fast Blue B, Fast Blue BB,Fast Corinth V, etc. are inherently unstable in solution, particularlyin aqueous solutions, and that this property has hindered thedevelopment of usable cannabinoid tests with this reagent, even forscreening purposes. Thus, in certain embodiments of the invention, thediazonium salt visualization reagent may be provided in its dry powderform, and in certain aspects may be pre-measured in a defined amount inthe reaction tube, or in a separate pouch to be added to the reactiontube. Other visualization reagents may similarly be provided in dryform. The user may then carry out the first steps of the quantificationassay method in a first tube, thus extracting the cannabinoids from asolid sample in a defined amount of extraction solvent composition andvolume, and then transfer a defined amount of the resultingcannabinoid-containing extraction liquid into a second tube containing adry, pre-measured visualization reagent, and then mixing to dissolve thevisualization reagent in the cannabinoid-containing extraction liquid,and comparing the resulting absorbance shift to a calibratedquantification reference chart. In the case of more than onevisualization reagent, each may be provided in separate tubes. Incertain embodiments of the invention, the diazonium salt may bestabilized as a salt of BF3, picric acid, sodium perchlorate, or othersalts known in the art to stabilize such diazonium compounds, forinstance as described in U.S. Pat. Nos. 4,771,005 and 8,124,420. Incertain embodiments of the invention, the dry reagent may also include asolid diluent, preferable an inert solid diluent. The solid diluent maybe useful for accurate measurement purposes, and may also serve as adessicant and/or light protectant to aid in the stability of the dryreagent. In certain embodiments, the solid diluent may be soluble in theextraction solution to be used in the visualization reaction.

In another aspect of the invention, there is provided a simplified assayfor quantification of CBD in a sample. Potassium hydroxide and sodiumhydroxide are cannabinoid-sensitive visualization reagents that arespecific for CBD. In general, to obtain an absorbance shift, thepotassium hydroxide must be contacted with CBD in an ethanolic medium.The inventors have disclosed herein that an absorbance shift may alsooccur with potassium hydroxide in a methanolic medium, or other loweralcoholic media. Thus, in order to reduce the number of steps for theCBD quantification assays of the present invention, it would bedesirable to extract the CBD from the sample using an extraction solventsuch as methanol, ethanol, propanol, or other lower alcohols. In thisway, the subsequent visualization reaction may be easily accomplished,by adding, for instance, solid potassium hydroxide in a pre-measuredamount, or by adding a small amount of a concentrated potassiumhydroxide solution, without a requirement to change the reaction medium,since the extracted CBD will be in a suitable solvent that is permissivefor the visualization reaction with potassium hydroxide. This simplifiedassay may be particularly suitable for development of a test kit, sincethe visualization reagent, for instance potassium hydroxide or sodiumhydroxide, may be provided in a non-alcoholic stock solution, such as anaqueous solution, that is more suitable for shipping and storage.Similarly, the visualization reagent, potassium hydroxide or sodiumhydroxide may be provided in a concentrated form, in either alcoholic ornon-alcoholic solution, thus allowing the addition of a very smallamount, such as a drop or a few drops, without requiring the CBDcontaining extraction solution to be exchanged to a lower alcohol toallow the visualization reaction.

Test Papers and Strips

Certain embodiments of the invention are based, in part, on thesurprising finding that test papers impregnated with certaincannabinoid-sensitive visualization reagents may be used to quantify thecannabinoid concentration in a sample. The inventor has providedexamples of such test papers suitable for quantifying cannabinoids suchas THC, CBD and/or CBN in a sample. Certain test papers are particularlyuseful for quantifying CBD in a test sample. Certain test papers areparticularly useful for quantifying THC in a test sample. In theseembodiments, the previously described methods for quantifyingcannabinoids using cannabinoid-sensitive visualization reagents isaccomplished by carrying out the visualization reaction on the testpaper or test strip, rather than in solution in a reaction vessel.

CBD Test Papers and Strips

In certain embodiments, the cannabinoid-sensitive visualization reagent,for instance KOH or NaOH, is specific for CBD, and is present on thetest paper in a sufficient amount to cause a colorimetric reaction onthe test paper when the test strip is contacted with CBD in a permissivesolvent. The permissive solvent may be an alcohol such as ethanol,methanol, or isopropanol. The inventors disclose that test stripsimpregnated with a CBD-specific cannabinoid-sensitive visualizationreagent such as KOH, which undergoes an absorbance shift (changes color)upon contact with CBD in an appropriate solvent, may be utilized toquantify the concentration of such CBD in a given sample. Quantificationis achieved by contacting the said test strip with a liquidCBD-containing sample (in a permissive solvent), or alternatively with aliquid extraction from a solid CBD-containing sample (in a permissivesolvent), and comparing the resulting absorbance shift to that caused bysamples with known CBD concentrations. The inventors have provided anexample of such an assay to determine the concentration of CBD in asolid plant sample. The inventors have provided an example of aquantification reference chart, useful in said assay, calibrated todetermine the CBD concentration in a solid plant sample, and suitablefor solid plant samples having CBD concentration between 0-15%, andpossibly higher. The calibrated reference chart has been optimized forthis CBD concentration range by extracting cannabinoids from a series ofsolid samples having known CBD concentrations ranging from 0% to 15%,using a uniform extraction solvent composition, volume, and extractiontime for all of the samples, and contacting the resulting CBD-containingextraction liquid with test strips impregnated with KOH. The resultingabsorbance shift, or color change, caused by each of the samples ofknown CBD concentration provides a reference chart for use in theexperimental assay for the unknown sample—by extracting the CBD from theunknown sample using the exact same extraction solvent composition,volume, and extraction time; contacting the resulting CBD-containingextraction liquid with the test papers impregnated with KOH; andcomparing the resulting absorbance shift, or color change, with thecalibrated quantification reference chart, one may thus determine theCBD concentration in the unknown solid sample. In an Example providedherein, the extraction solvent composition, volume, and extraction timehave been optimized to ensure that the absorbance shift caused bycontacting the resulting CBD-containing extraction solution with testpapers impregnated with KOH is in the linear range of the visualizationreagent, such that the absorbance, or color change intensity, isproportional to the CBD concentration. An important feature of thequantification method using the KOH-impregnated test papers is that thesolvent used to extract or otherwise dissolve the CBD is of anappropriate composition to allow the visualization reaction to occur onthe test paper. In an Example detailed herein, for instance, the KOH isdried onto the paper, and the CBD-containing test samples are dissolvedin methanol or propanol, thus when the liquid test sample is contactedwith the test strip, the methanol or propanol solvent creates anappropriate environment for the visualization reaction to occur betweenthe KOH and CBD. In these various embodiments, the test papers may beaffixed to a solid support, for instance to form a test strip,colorimetric strip, dipstick, and the like. As used herein, the terms‘test paper’ and ‘test strip’ may be used interchangeably.

In one aspect of the invention, there is provided an apparatus includinga test strip wherein the test strip comprises a porous matrix uniformlyimpregnated with a CBD-specific cannabinoid sensitive visualizationreagent. The CBD-specific cannabinoid-sensitive visualization reagentmay be a strong base. The CBD-specific cannabinoid-sensitivevisualization reagent may be KOH or NaOH. In certain embodiments, theCBD-specific cannabinoid-sensitive visualization reagent is present onthe test strip in sufficient amount to cause a colorimetric chemicalreaction when contacted with CBD and a permissive solvent. Thepermissive solvent may be an alcohol. The permissive solvent may beethanol, methanol, or propanol. In some embodiments, the CBD-specificcannabinoid-sensitive visualization reagent is present in the test paperin sufficient amount to allow quantification of CBD in a CBD containingsample.

In another aspect of the invention, there is provided a method forquantification of the concentration of CBD in a liquid sample, themethod involves: 1) contacting the liquid sample with a test stripcomprising a porous matrix uniformly impregnated with a CBD-specificcannabinoid-sensitive visualization reagent, wherein the liquid samplecomprises a solvent of an appropriate composition to allow acolorimetric reaction between the CBD and the CBD-specificcannabinoid-sensitive visualization reagent; 2) removing the test stripfrom the liquid sample and allowing it to develop for a defined amountof time; and 3) comparing the intensity of the resulting color change,or absorbance shift, of the test strip to a calibrated quantificationreference chart, such that comparison of the color intensity change orabsorbance shift of the test sample to calibrated quantificationreference chart allows determination of the CBD concentration in thetest sample. The calibrated quantification reference chart may beproduced, for instance, by contacting a series of calibrated sampleshaving pre-determined CBD concentrations with a series of test stripsunder the exact same conditions as to be used for the test sample,including using the same CBD-specific cannabinoid-sensitivevisualization reagent, the same liquid solvent, and the same colordevelopment time as to be used for the test sample. The CBD-specificcannabinoid-sensitive visualization reagent may be a strong base. TheCBD-specific cannabinoid-sensitive visualization reagent may be a KOH.The permissive liquid solvent may be an alcohol. The appropriate liquidsolvent may be methanol, ethanol, or isopropanol.

In another aspect of the invention, there is provided a method forquantification of CBD in a solid sample, the method involves: 1)contacting a defined amount of solid sample with a defined volume of anextraction solvent for a defined amount of time, wherein CBD isextracted from the solid sample into the extraction solvent resulting ina CBD-containing liquid extraction solution, and wherein the extractionsolvent has a composition suitable to allow the reaction of theextracted CBD with a CBD-specific cannabinoid-sensitive visualizationreagent; 2) contacting the resulting liquid extraction solution with atest strip comprising a porous matrix uniformly impregnated with aCBD-specific cannabinoid-sensitive visualization reagent; 3) removingthe test strip from the liquid extraction solution and allowing it todevelop for a defined amount of time; and 4) comparing the intensity ofthe resulting color change, or absorbance shift, of the test strip to acalibrated quantification reference chart, such that comparison of thecolor intensity change or absorbance shift of the test sample to acalibrated quantification reference chart allows determination of theCBD concentration in the test sample. The calibrated quantificationreference chart may be created by performing the identical method asused on the unknown solid sample on a series of solid samples of knownCBD concentration, and recording the resultant color change, orabsorbance shift, caused by the known CBD concentrations. TheCBD-sensitive visualization reagent may be a strong base. TheCBD-specific cannabinoid-sensitive visualization reagent may be a KOH.The extraction solvent may be an alcohol. The extraction solvent may bemethanol, ethanol, or isopropanol.

The inventors further disclose that test papers or strips impregnatedwith a strong base such as KOH rapidly lose effectiveness towards CBDwhen exposed for only a short time to ambient air. This means that suchstrips may only be effective when freshly prepared, which is asignificant limitation for the applicability in field tests or in kits.The inventors have successfully extended the shelf life of such teststrips by vacuum sealing to remove any air and/or moisture fromcontacting the strips, allowing their use days, weeks, or months afterpreparation. In certain embodiments, there is provided a CBD-sensitivetest paper and/or strip comprising a porous matrix uniformly impregnatedwith a CBD-sensitive visualization reagent, wherein the test paperand/or strip is provided in a sealed container or package. The sealedcontainer or package may include a dessicant.

THC Test Papers and Strips

The inventors also provide examples of compositions and methods forquantifying cannabinoids THC, CBD and/or CBN in a test sample, usingtest strips comprising a porous matrix impregnated withcannabinoid-sensitive visualization reagents. In certain embodiments ofthe invention, the test strip comprises multiple test papers whereineach test paper is impregnated with a different cannabinoid-sensitivevisualization reagent.

In other aspects of the invention, the one or more cannabinoid-sensitivevisualization reagents is a diazonium salt. In other embodiments, one ofthe cannabinoid-sensitive visualization reagents is a Ghamrawy reagentor a modified Ghamrawy reagent. The inventors have developed a methodfor manufacturing test papers with a modified Ghamrawy reagent. Suchreagents have been described as requiring hydrochloric acid or sulfuricacid to be effective. As these acids are generally only present inliquid form, these reagents have previously only been suitable forliquid based reactions. The inventors have identified strong acids thatmay also exist as a solid, for instance p-toluenesulfonic acid. Thus, incertain embodiments, the modified Ghamrawy reagent comprises p-DMAB andp-toluenesulfonic acid. In certain embodiments, the test paper ismanufactured by dissolving the modified Ghamrawy reagents in a solventsuch as methanol—either together or separately—and then contacting thetest paper with the reagent and allowing to dry. The test strip may beheated during the drying process, which the inventors have shown toimprove the effectiveness of the test papers. In other embodiments, thetest strips may comprise a Duquenois reagent.

In other aspects of the invention, there is thus provided an apparatuswhich includes a test strip, wherein the test strip comprises a porousmatrix uniformly impregnated with one or more cannabinoid-sensitivevisualization reagents. In certain embodiments, the one or morecannabinoid-sensitive visualization reagents are present on the teststrip in sufficient amount to cause a colorimetric chemical reactionwhen contacted with cannabinoids and an appropriate solvent. In someembodiments, the cannabinoid-sensitive visualization reagents arepresent in sufficient amount to allow quantification of THC and/or CBDand/or CBN in a cannabinoid containing sample. In certain embodiments,the one or more visualization reagents are present in different discreteregions of the test strip, for instance on separate test papers that areaffixed to the test strip. In certain embodiments, thecannabinoid-sensitive visualization reagents are diazonium salts such asFast Blue B, Fast Blue BB, Fast Garnet, and/or Fast Corinth V. Incertain embodiments, the cannabinoid-sensitive visualization reagent mayalso be a Duquenois reagent. In certain embodiments, thecannabinoid-sensitive visualization reagent may be a Ghamrawy reagent ora modified Ghamrawy reagent. In certain embodiments, the test stripfurther comprises an inert support.

In another aspect of the invention, there is provided a method forquantification of the concentration of one or more cannabinoid compoundsin a liquid sample, the method involves: 1) contacting the liquid samplewith a test strip comprising a porous matrix uniformly impregnated withone or more cannabinoid-sensitive visualization reagents; 2) removingthe test strip from the liquid sample and allowing it to develop for adefined amount of time; and 3) comparing the intensity of the resultingcolor change, or absorbance shift, of the one or morecannabinoid-sensitive visualization reagents to a calibratedquantification reference chart, such that comparison of the colorintensity change or absorbance shift of the test sample to thecalibrated quantification reference chart allows determination of thecannabinoid concentration in the test sample. The calibratedquantification reference chart may be produced, for instance, bycontacting a series of calibrated samples having pre-determinedcannabinoid concentrations with a series of test strips under the exactsame conditions as to be used for the test sample, including using thesame one or more cannabinoid-visualization reagents and the same colordevelopment time as to be used for the test sample. In certainembodiments, the test strip comprises one or more test papers, eachindividually impregnated with different cannabinoid-sensitivevisualization reagents. The cannabinoid-sensitive visualization reagentmay be a diazonium salt. The cannabinoid-sensitive visualization reagentmay be chosen from the following: Fast Blue B, Fast Blue BB, Fast Garnetand Fast Corinth V. The cannabinoid-sensitive visualization reagent maybe a Duquenois reagent. The cannabinoid compound to be quantified may beTHC. In certain embodiments, the cannabinoid-sensitive visualizationreagent may be a Ghamrawy reagent or a modified Ghamrawy reagent.

In another aspect of the invention, there is provided a method forquantification of one or more cannabinoid compounds in a solid sample,the method involves: 1) contacting a defined amount of solid sample witha defined volume of an extraction solvent for a defined amount of time,wherein cannabinoids are extracted from the solid sample into theextraction solvent resulting in a cannabinoid-containing liquidextraction solution; 2) contacting the resulting liquid extractionsolution with a test strip comprising a porous matrix uniformlyimpregnated with one or more cannabinoid-sensitive visualizationreagents; 3) removing the test strip from the liquid extraction solutionand allowing it to develop for a defined amount of time; and 4)comparing the intensity of the resulting color change, or absorbanceshift, of the cannabinoid-sensitive visualization reagent to acalibrated quantification reference chart, such that comparison of thecolor intensity change or absorbance shift of the test sample to acalibrated quantification reference chart allows determination of thecannabinoid concentration in the test sample. The calibratedquantification reference chart may be created by performing theidentical method as used on the unknown solid sample on a series ofsolid samples of known cannabinoid concentration, and recording theresultant color change, or absorbance shift, caused by the knowncannabinoid concentrations. In certain embodiments, the test stripcomprises one or more test papers, each individually impregnated withdifferent cannabinoid-sensitive visualization reagents. Thecannabinoid-sensitive visualization reagent may be a diazonium salt. Thecannabinoid-sensitive visualization reagent may be chosen from thefollowing: Fast Blue B, Fast Blue BB, Fast Garnet and Fast Corinth V.The cannabinoid compound to be quantified may be THC. Thecannabinoid-sensitive visualization reagent may be a Duquenois reagent.The cannabinoid compound to be quantified may be THC. In certainembodiments, the cannabinoid-sensitive visualization reagent may be aGhamrawy reagent or a modified Ghamrawy reagent

Kits

In another aspect of the invention, there is provided a kit forquantification of the concentration of one or more cannabinoid compoundsin a sample, the kit comprising: 1) optionally, a solvent for extractionof cannabinoid compounds from a solid sample; 2) one or morecannabinoid-sensitive visualization reagent; and 3) a calibratedquantification reference chart. In a further embodiment, the calibratedquantification reference chart may be replaced by an instruction or setof instructions guiding or directing to a calibrated quantificationreference chart, for instance via web link, URL, email address, or othermeans. In certain embodiments, the extraction solvent has a compositionthat is permissive for a visualization reaction with thecannabinoid-sensitive visualization reagents. In certain embodiments,the kit is useful for the quantification of THC in a test sample, andthe one or more cannabinoid-sensitive visualization reagents is chosenfrom: a diazonium salt, for instance Fast Blue B, Fast Blue BB, FastCorinth V, and Fast Garnet GC; a Duquenois reagent; a Ghamrawy reagent;and/or a modified Ghamrawy reagent. In certain embodiments of theinvention the kit is useful for the quantification of CBD in a testsample, and the cannabinoid-sensitive visualization reagent is a strongbase, for instance potassium hydroxide or sodium hydroxide. In certainembodiments, the cannabinoid-sensitive visualization reagent is providedin a pre-measured amount suitable for single test reactions. In certainembodiments, the pre-measured cannabinoid-sensitive visualizationreagent is provided in dry form. The pre-measured cannabinoid-sensitivevisualization reagent may be provided in a ready to use reaction vessel,for instance a test tube, plastic tube, eppendorf tube, and the like, orin a separate pouch or container. In certain embodiments, the dryreagent may further include a solid diluent. In certain embodiments, theextraction solvent may have a composition that is: 1) suitable fordissolving the dry pre-measured cannabinoid-sensitive visualizationreagent; and 2) permissive for a visualization reaction between thecannabinoid and the cannabinoid-sensitive visualization reagent.

In another aspect of the invention, there is provided a kit for extendedrange quantification of the concentration of one or more cannabinoidcompounds in a sample, the kit includes: 1) optionally, a solvent forextraction of cannabinoid compounds from the sample; 2) two or morecannabinoid-sensitive visualization reagents; and 3) a calibratedquantification reference chart, wherein the calibrated quantificationreference chart comprises calibrated quantification reference for eachof the two or more cannabinoid-sensitive visualization reagents. In afurther embodiment, the calibrated quantification reference chart may bereplaced by an instruction or set of instructions guiding or directingto a calibrated quantification reference chart, for instance via weblink, URL, email address, or other means. In certain embodiments, theextraction solvent has a composition that is permissive for avisualization reaction with the two or more cannabinoid-sensitivevisualization reagents. In certain embodiments, the kit is useful forthe quantification of THC in a test sample, and thecannabinoid-sensitive visualization reagents are chosen from: adiazonium salt, for instance Fast Blue B, Fast Blue BB, Fast Corinth V,and Fast Garnet GC; a Duquenois reagent; a Ghamrawy reagent; and/or amodified Ghamrawy reagent. In certain embodiments, thecannabinoid-sensitive visualization reagent is provided in apre-measured amount suitable for a single test reaction. In certainembodiments, the pre-measured cannabinoid-sensitive visualizationreagent is provided in dry form. The dry reagent may further include asolid diluent The pre-measured cannabinoid-sensitive visualizationreagent may be provided in a ready to use reaction vessel, for instancea test tube, plastic tube, eppendorf tube, and the like, or may beprovided in a separate pouch or container. In certain embodiments, theextraction solvent may have a composition that is 1) suitable fordissolving the dry pre-measured cannabinoid-sensitive visualizationreagent; and 2) permissive for a visualization reaction between thecannabinoid and the cannabinoid-sensitive visualization reagent.

The following examples are provided for illustrative purposes, and arenot intended to be limiting.

EXAMPLES Example 1 Quantification of THC Content of an Unknown SolidCannabis Sample Using the Compositions and Methods of theInvention—Visualization in Solution

Preparation of Cannabinoid-Sensitive Visualization Reagents

Fast Blue BB diazonium salt was dissolved in methanol to a finalconcentration of 0.1%.

Production of Calibrated Quantification Reference Chart

Solid cannabis samples with known THC concentration were used to createthe calibrated quantification reference chart. These cannabis sampleswere known to have negligible concentrations of other cannabinoids thatmight also react with the cannabinoid-sensitive visualization reagent,such as CBD or CBN. For each sample, the following protocol was followedto extract the THC into a liquid THC containing solution. The solidsample was macerated into small pieces, and a portion was wrapped inaluminium foil. This was heated at 325° F. for exactly 5 minutes. Theresulting solid sample was removed from the foil, crumbled, and a 0.1 gportion was placed into a plastic tube. Exactly 5.0 mL of methanol orisopropanol was placed into the plastic tube. The tube with the 0.1 gsolid sample and 5.0 mL of methanol or isopropanol was shaken vigorouslyfor 30 seconds. A 1.0 mL aliquot of the resulting THC containingextraction solution was transferred to an eppendorf tube. One drop (˜20uL) of cannabinoid-sensitive visualization reagent from step A wasadded, and the reaction was allowed to develop for exactly 10 minutes.This procedure was completed in parallel with a number of solid cannabissamples having different known THC concentrations. After the colourdevelopment step, all the resulting color reactions were used to createthe calibrated quantification reference chart shown in FIG. 1 . Thecolors from each tube are then easily transferred to a suitable medium,for instance onto a reference card and the like, for ease of comparisonat any future point. More specifically, as shown in FIG. 1 , thecalibrated quantification reference chart for THC samples is disclosedhaving an ideal range of 5-20%. The quantification reference chart wasgenerated using Fast Blue BB. The chart shows a light yellow colour atthe left side, changing to a darker yellow/orange on the right side.

A. Determination of THC Concentration in Unknown Solid Cannabis Sample

A solid cannabis sample with unknown THC concentration (and known tohave low concentration of other cannabinoids that might react with thecannabinoid-sensitive visualization reagent, such as CBD or cannabinol)was prepared using the exact same methodology as the samples in Step B.The resulting THC-containing extraction liquid was contacted withcannabinoid-sensitive visualization reagent from Step A and colordevelopment for 10 minutes, exactly as in Step B. The resulting colorchange was then compared to the calibrated quantification referencechart from Step B to determine the THC concentration in the unknownsample.

Example 2 Quantification of THC Content of an Unknown Solid CannabisSample Using an Extended Range Calibrated Quantification ReferenceChart—Visualization in Solution

A. Preparation of Two Cannabinoid-Sensitive Visualization Reagents

Fast Blue BB salt was dissolved in methanol to a final concentration of0.1%. Fast Corinth V was dissolved in methanol to a final concentrationof 0.1%

B. Production of Calibrated Quantification Reference Chart

Solid cannabis samples with known THC concentration were used to createthe calibrated quantification reference chart. These cannabis sampleswere known to have negligible concentrations of other cannabinoids thatmight also react with the cannabinoid-sensitive visualization reagent,such as CBD or cannabinol. For each sample, the following protocol wasfollowed to extract the THC into a liquid THC containing solution. Thesolid sample was macerated into small pieces, and a portion was wrappedin aluminium foil. This was heated at 325 degrees Celsius for exactly 5minutes. The resulting solid sample was removed from the foil, crumbed,and a 0.1 g portion was placed into a plastic container. Exactly 5.0 mLof methanol or isopropanol was placed into the plastic container. Thecontainer with the 0.1 g solid sample and 5.0 mL of methanol orisopropanol was shaken vigorously for 10 seconds. A 1.0 mL aliquot ofthe resulting THC containing extraction solution was transferred to afirst eppendorf tube, and a 1.0 mL aliquot of the resulting THCcontaining extraction solution was transferred to a second eppendorftube. One drop (˜20 uL) of Fast Blue BB cannabinoid-sensitivevisualization reagent from step A was added to the first eppendorf tube,and one drop (˜20 uL) of Fast Corinth V cannabinoid-sensitivevisualization reagent from step A was added to the second eppendorftube, and the reaction in each tube was allowed to develop for exactly10 minutes. This procedure was completed in parallel with a number ofsolid cannabis samples having different known THC concentrations. Afterthe colour development step, all the resulting color changes for FastBlue BB, and the resulting color changes for Fast Corinth V were used tocreate the extended range calibrated quantification reference chart(FIG. 2 ). More specifically, as shown in FIG. 2 , an extended rangecalibrated quantification reference chart is detailed therein. The chartincludes overlapping ranges for 2 different cannabinoid-sensitivevisualization reagents, to allow for quantification from 0% to over 25%THC in a solid sample. The top row of colors is generated using FastCorinth V. The bottom row of colors is generated using Fast Blue BB. Thetop row, moving from left to right, shows the color change starting aslight orange at the bottom of the range and getting progressively morered at the top of the range. The ideal quantification range for the toprow is 0-14%. The bottom row, moving from left to right, shows the colorchange starting at light yellow at the bottom of the range, and movingto darker orange at the top of the range. The ideal quantification rangeis 11-25%. The overlapping ideal ranges provides good quantificationbetween 0-25% THC.

C. Determination of THC Concentration in Unknown Solid Cannabis Sample

A solid cannabis sample with unknown THC concentration (and known tohave low concentration of other cannabinoids that might react with thecannabinoid-sensitive visualization reagent, such as CBD or cannabinol)was prepared using the exact same methodology as the samples in Step B.The resulting THC-containing extraction liquid was contacted withcannabinoid-sensitive visualization reagent from Step A and colourdevelopment for 10 minutes, exactly as in Step B. The resulting colourchange was then compared to the extended range calibrated quantificationreference chart from Step B to determine the THC concentration in theunknown sample.

Example 3 Quantification of CBD Content of an Unknown Solid CannabisSample Using the Compositions and Methods of the Invention—Visualizationin Solution

A. Preparation of Cannabinoid-Sensitive Visualization Reagent

Potassium hydroxide was dissolved in water to a final concentration of20%.

B. Production of Calibrated Quantification Reference Chart

Solid cannabis samples with known CBD concentration were used to createthe calibrated quantification reference chart. For each sample, thefollowing protocol was followed to extract the CBD into a liquid CBDcontaining solution. The solid sample was macerated into small pieces,and a portion was wrapped in aluminium foil. This was heated at 325degrees Celsius for exactly 5 minutes. The resulting solid sample wasremoved from the foil, crumbed, and a 0.1 g portion was placed into aplastic container. Exactly 3.0 mL of methanol or isopropanol was placedinto the plastic container. The container with the 0.1 g solid sampleand 3.0 mL of methanol or isopropanol was shaken vigorously for 10seconds. A 1.0 mL aliquot of the resulting THC containing extractionsolution was transferred to an eppendorf tube. Two drops (˜40 uL) ofcannabinoid-sensitive visualization reagent potassium hydroxide fromstep A was added, and the reaction was allowed to develop for exactly 10minutes. This procedure was completed in parallel with a number of solidcannabis samples having different known CBD concentrations. After thecolour development step, all the resulting reactions were lined up inorder of increasing THC concentration and the colors used to create thecalibrated quantification reference chart (FIG. 3 ). The colors fromeach tube are then easily transferred to a suitable medium, for instanceonto a reference card and the like, for ease of comparison at any futurepoint. More specifically, as shown in FIG. 3 , a calibratedquantification reference chart for CBD samples is depicted demonstratingan ideal range of 0-15%. Moving from left to right, the figure shows thecolor changing from very light purple at the lower end of the scale to amuch darker purple at the higher end of the scale.

C. Determination of THC Concentration in Unknown Solid Cannabis Sample

A solid cannabis sample with unknown CBD concentration was preparedusing the exact same methodology as the samples in Step B. The resultingCBD-containing extraction liquid was contacted withcannabinoid-sensitive visualization reagent from Step A and colourdevelopment for 10 minutes, exactly as in Step B. The resulting colourchange was then compared to the extended range calibrated quantificationreference chart from Step B to determine the CBD concentration in theunknown sample.

Example 4 Quantification of THC Content of an Unknown Solid CannabisSample Using the Compositions and Methods of the Invention

A. Preparation of Test Strips Impregnated with Fast Blue BB

Fast Blue BB salt was dissolved in methanol to a final concentration of1%. Strips of Whatman paper (CF2) were submersed in the 1% Fast Blue BBmethanol solution, removed, and allowed to dry.

B. Production of Calibrated Quantification Reference Chart

Solid cannabis samples with known THC concentration were used to createthe calibrated quantification reference chart. For each sample, thefollowing protocol was followed to extract the THC into a liquid THCcontaining solution. The solid sample was macerated into small pieces,and a portion was wrapped in aluminium foil. This was heated at 325degrees Celsius for exactly 5 minutes. The resulting solid sample wasremoved from the foil, crumbed, and a 0.1 g portion was placed into aplastic container. Exactly 15 mL of methanol or isopropanol was placedinto the plastic container. The container with the 0.1 g solid sampleand 15 mL of methanol or isopropanol was shaken vigorously for 10seconds. A 1.5 mL aliquot of the resulting THC containing extractionsolution was transferred to an eppendorf tube. An unused Fast Blueimpregnated test strip from Part A was dipped briefly into the THCcontaining extraction solution, and excess liquid was shaken off thetest strip. The test strip was allowed to air dry and the colour wasallowed to develop for exactly 10 minutes from the point where the stripfirst touched the THC containing extraction solution. This procedure wascompleted in parallel with a number of solid cannabis samples havingdifferent known THC concentrations. After the colour development step,all the resulting test strips were lined up in order of increasing THCconcentration, and a picture taken to create the calibratedquantification reference chart—see FIG. 4 . More specifically, as shownin FIG. 4 , a calibrated reference chart for Fast BB impregnated teststrips is depicted. The chart shows that, moving from left to right, thecolor change of the strip goes from a very light orange/red at the lowend of the scale to a much darker red at the higher end of the scale.

C. Determination of THC Concentration in Unknown Solid Cannabis Sample

A solid cannabis sample with unknown THC concentration was preparedusing the exact same methodology as the samples in Step B. The resultingTHC-containing extraction liquid was tested by dipping a Fast Blue BBimpregnated test strip from Step A briefly into the THC containingextraction liquid, removing excess liquid, and allowing air drying andcolour development for 10 minutes, exactly as in Step B. The resultingcolour change was then compared to the calibrated quantificationreference chart from Step B to determine the THC concentration in theunknown sample.

Example 5 Quantification of CBD Content of an Unknown Solid CannabisSample Using the Compositions and Methods of the Invention

A. Preparation of Test Strips Impregnated with KOH

KOH was dissolved in methanol to a final concentration between 1-30%methanol (1%, 10%, 30%). Strips of Whatman paper (CF1) were submersed inthe KOH solution, removed, and allowed to dry. Dried test strips weredipped in a methanol extract of a CBD containing Cannabis sample. TheCannabis sample contained 15% CBD and the extract was prepared by i)macerating the sample and heating at 325 degrees Fahrenheit for 5minutes to convert CBD-A to CBD; ii) adding 0.1 g of the heated sampleto 1.0 mL of methanol; and iii) shaking vigorously for 20 seconds. Afterthe test strips were dipped in the CBD liquid extract, they were allowedto develop for 5 minutes. FIG. 5 shows that impregnation of the teststrips using 1% or 10% KOH in methanol did not result in a significantcolor change in the subsequent reaction with the CBD liquid extract,however using 30% did allow for a significant colorimetric reaction tooccur. More specifically, as shown in FIG. 5 , representative testpapers are depicted having been prepared using 30% KOH (left), 10% KOH(middle), and 1% KOH (right).

B. Production of Calibrated Quantification Reference Chart

Solid cannabis samples with known CBD concentration were used to createthe calibrated quantification reference chart. For each sample, thefollowing protocol was followed to extract the CBD into a liquid CBDcontaining solution. The solid sample was macerated into small pieces,and a portion was wrapped in aluminium foil. This was heated at 325degrees Fahrenheit for exactly 5 minutes. The resulting solid sample wasremoved from the foil, crumbled, and a 0.1 g portion was placed into aplastic container. Exactly 1.0 mL of methanol or isopropanol was placedinto the plastic container. The container with the 0.1 g solid sampleand 1.0 mL of methanol or isopropanol was shaken vigorously for 20seconds. An aliquot of the resulting CBD containing extraction solutionwas transferred to an eppendorf tube. A KOH impregnated test strip wasprepared as in Part A, except that the impregnation of the test stripswas performed with KOH dissolved in methanol at a concentration of 20%w/v. An unused KOH impregnated test strip was dipped briefly into theCBD containing extraction solution, and excess liquid was shaken off thetest strip. The test strip was allowed to air dry and the color wasallowed to develop for exactly 5 minutes from the point where the stripfirst touched the CBD containing extraction solution. This procedure wascompleted in parallel with a number of solid cannabis samples havingdifferent known CBD concentrations. After the color development step,all the resulting test strips were lined up in order of increasing CBDconcentration, and a picture taken to create the calibratedquantification reference chart—see FIG. 6 . It can be seen that thecolor intensity increases in direct correlation with the increasing CBDconcentration in the sample. More specifically, as shown in FIG. 6 , aCBD Calibrated Reference Chart is depicted. Moving from left to right onthe chart therein, the color change shown on the chart goes from a verylight violet/purple for the lower CBD % to a much darker purple forhigher CBD %.

C. Determination of CBD Concentration in Unknown Solid Cannabis Sample

A solid cannabis sample with unknown CBD concentration was preparedusing the exact same methodology as the samples in Step B. The resultingCBD-containing extraction liquid was tested by dipping a KOH impregnatedtest strip from Step A briefly into the CBD containing extractionliquid, removing excess liquid, and allowing air drying and colordevelopment for 5 minutes, exactly as in Step B. The resulting colorchange was then compared to the calibrated quantification referencechart from Step B to determine the CBD concentration in the unknownsample.

Example 6 Quantification of THC Content of an Unknown Solid CannabisSample Using the Compositions and Methods of the Invention

A. Preparation of Test Papers Impregnated with Modified Ghamrawy Reagent

A 10% solution of p-dimethylaminobenzaldehyde (p-DMAB) in methanol wasprepared in a tube. In a separate tube, a 5M solution ofp-toluenesulfonic acid in methanol was prepared. Squares of CF4 paper(GE Healthcare) were immersed in the p-DMAB solution and allowed to dry,and then quickly dipped in the p-toluenesulfonic acid solution. Thestrips were then placed in an oven at 200 degrees Fahrenheit for 2minutes until the strips were completely dry.

B. Preparation of Calibrated Reference Chart

Solid cannabis samples with known THC concentration were used to createthe calibrated quantification reference chart. For each sample, thefollowing protocol was followed to extract the THC into a liquid THCcontaining solution. The solid sample was macerated into small pieces,and a portion was wrapped in aluminium foil. This was heated at 325degrees Celsius for exactly 5 minutes. The resulting solid sample wasremoved from the foil, crumbed, and a 0.1 g portion was placed into aplastic container. Exactly 15 mL of methanol or isopropanol was placedinto the plastic container. The container with the 0.1 g solid sampleand 15 mL of methanol or isopropanol was shaken vigorously for 10seconds. A 1.5 mL aliquot of the resulting THC containing extractionsolution was transferred to an eppendorf tube. An unused modifiedGhamrawy reagent impregnated test strip from Part A was dipped brieflyinto the THC containing extraction solution, and excess liquid wasshaken off the test strip. The test strip was allowed to air dry and thecolour was allowed to develop for exactly 10 minutes from the pointwhere the strip first touched the THC containing extraction solution.This procedure was completed in parallel with a number of solid cannabissamples having different known THC concentrations. After the colourdevelopment step, all the resulting test strips were lined up in orderof increasing THC concentration, and a picture taken to create thecalibrated quantification reference chart—see FIG. 7 . Morespecifically, as shown in FIG. 7 , a THC Calibrated Reference Chartusing modified Ghamrawy reagent impregnated test papers is depicted.Moving from left to right on the chart therein, the color change shownon the chart goes from a very light violet/purple for the lower THC % toa much darker purple for higher THC %.

C. Quantification of THC Concentration in a Test Sample of UnknownConcentration

A solid cannabis sample with unknown THC concentration (and known tohave low concentration of other cannabinoids that might react with thecannabinoid-sensitive visualization reagent, such as CBD) was preparedusing the exact same methodology as the samples in Step B. The resultingTHC-containing extraction liquid was contacted withcannabinoid-sensitive visualization reagent from Step A and colourdevelopment for 10 minutes, exactly as in Step B. The resulting colourchange was then compared to the calibrated quantification referencechart from Step B to determine the THC concentration in the unknownsample.

While specific embodiments of the invention have been described andillustrated, such embodiments should be considered illustrative of theinvention only and not as limiting the invention as construed inaccordance with the accompanying claims. Other features and advantagesof the invention will be apparent from the following description of thedrawings and the invention, and from the claims.

What is claimed is:
 1. A method for quantification of the concentrationof one or more cannabinoid compounds in a liquid sample, the methodcomprising: a) contacting the liquid sample with at least onecannabinoid-sensitive visualization reagent; b) allowing the at leastone cannabinoid-sensitive visualization reagent to develop for a definedamount of time; and c) comparing the resulting color change of the atleast one cannabinoid-sensitive visualization reagent to a calibratedquantification reference chart.